DE2360118A1 - VACCINES AGAINST PIGLET COLIBACILLOSIS AND THE PROCESS FOR THEIR PRODUCTION - Google Patents

Description

RECHERCHE ET INDUSTRIE TIIERAPEUTIQUES, R .. Ί.T., Genval, Belgium

"Vaccines Against Piglet Colibacillosis and -Procedures-to Their Manufacturing "·

Priority: December 4, 19? 2, V.Ot./u, Kr. 511 998.

The invention relates to new vaccines gogen oolibacillose, in particular Vaccines against piglet golibacillosis.

The neonatal colibacillosis of the Schv / eins is an important death cause of "piglets. Numerous attempts have already been made v / orden to avoid this disease by actively immunizing the Dams against Eseherichia coli either by dead or Live vaccines or by administering Eseherichia coli-Anti Seriün to the Schv.'eine to b

Active immunization; the sows iat by parenteral Vsrabreichuiif; of Eseherichia eoli vaccines. (see e.g. U.R. VJiI-spri and J * Svendsen, Amer, J. Vet. Res., 'Bond J2 (1973), pp

to 89S) made v / ordon. l> a, however, the or those for Ι-ητπι-verv.'eiideten Eseherichia coli strains only specific 409823/1134

Elicit antibodies, while multiple and different strains may be associated with a particular outbreak of the disease, these attempts at prevention based on antibacterial immunity have been contradicting and generally unsatisfactory Results. Even when good protection was obtained in some cases, it was on the serotypes contained in the vaccine restricted by Escherichia coli.

The administration of antiserum (see e.g. O.P. Miniats. Can. Vet, J., Volume 11 (1970), pages 52 to 56) cannot be a practicable prevention ensure against piglet oleibacillosis. The protection achieved by the administration of antiserum is actually from short duration and requires continuous administration to the piglets.

It has now surprisingly been found that when administered of heat labile (LT) Escherichia coli enterotoxin with an adjuvant to pregnant sows 3 to 6 weeks before piglets instead of giving whole cells of Escherichia coli, the vaccinated sows not only have antibodies in their colostrum

and in their .. milk develop, but also the piglets be protected against the effects of interotoxins. The protection extends against the effects of snterotoxins caused by Escherichia coli serotypes are formed which can be both homologous and heterologous to the Escherichia coli strain from which the heat-labile (LT) enterotoxin was isolated. In other words, the administration of the heat labile (LT) Escherichia coli enterotoxin protects to the dam the piglets not only against the heat-labile (LT) Sscherichia coli enterotoxin caused

409823/1134 9ADOR ₁ GINAL

Colibacillosei but also against the hitaestabile (ST) Enterotoxin caused colibacillosis.

The object of the "invention was therefore to develop new vaccines against piglet coliforms to provide with which this disease can be reliably combated. This task is carried out by solved the invention.

The invention thus relates to vaccines against piglet colibacillosis, which contain an effective amount of cell-free, heat-labile (LT) Escherichia eoli enterotoxin and an adjuvant.

The aforementioned heat-labile (LT) Escherichia coli enterotoxin can be isolated from any enteropathogenic Escherichia coli serotype v / earth, which can cause piglet colibacillosis and necessarily the heat-labile (LT) Escherichia coli enterotoxin contains. A preferred example of such an enteropathogenic scherichia coli gerotype is that in American Type Culture Collection, Rockville, Maryland, T.St.A. under the lobster ATCC 21 972 deposited Escherichia coli strains.

The isolation of the heat labile (LS) Escherichia coli enterotoxin is known. It has been described by GYLE3 and BARNUH (J. Infect. Dis., Volume 120 (1969), pages 419 to 426); Variations of this procedure are 'von HV /. SMITH and CL. GYLES (J.. I-Ied. Mcrobiol., Vol. 3 (1970), pages 387 to 401), HV '. SMITH and S. HALLS (J. rath. Bact., Volume 93 (196?), Pages 531 to 543) and KH · './ILOON, G. GYLES and J. SVEIiIVJE ^ (Cand. J. Comp. Med. , Volume 35 (1971), pages 294 to 297). All of these methods and all other, known- .equivalenbe V _f er ^ .-

409823/1134. \

8A ORIGINAL '

drive can be used to produce the heat-labile (LT) Escherichia coli enterotoxin preparation according to the invention.

It is not necessary for the present invention to produce the heat-labile (LT) Escherichia coli enterotoxin in pure form. Such purification could, if necessary, e.g. by electrophoresis, density gradient ultracentrifugation or adsorption / Elution on hydrophilic crosslinked dextrans, v / ie Sephadex (one manufactured by Pharmacia Fine Chemicals AB, Uppsala I, Sweden and distributed product). For the present invention, it is essentially sufficient that the Enterotoxin released and separated from the Escherichia coli cells and is practically free of lipopolysaccharides.

As an adjuvant that has the task of creating a strong immune response can ensure, according to the invention, any product or mixture which is known to be used as an adjuvant in vaccines works. Specific examples of adequate adjuvants are gel-like aluminum compounds such as aluminum hydroxide and aluminum phosphate and ALHYDROGEL (trademark of one of SUPERFOS Export Company, Copenhagen, manufactured and sold aluminum hydroxide gels), and water-in-oil emulsions such as that Adjuvant 65 (a water-in-oil emulsion of antigen in peanut oil, that emulsified by mannitol monooleate and by aluminum monostearate stabilized) and complete Freund's adjuvant (a water-in-oil emulsion of paraffin oil emulsified with mannitol monooleate and killed Ilycobacterium tuberculosis contains).

v / is an effective amount of the heat labile (LT) 409823/1134

Escherichia coli enterotoxin preparation with the adjuvant to pregnant women Sows administered intramuscularly or subcutaneously. The dosage unit is at least 5 mg and preferably 10 to 150 mg freeze-dried extract.

• The examples explain the invention.

Example 1 .

Growing medium (solid):

g BACTO-TRYPTOSE broth and I5Q g BACTO-AGAR (BACT0 - TRYPTQ3E and BAGTO-AGAR are trade names for products made by the DIFCO Laboratories, Detroit 1, Michigan, V.St.A. to be expelled) 10 liters of water are added. The mixture is heated to 121 ° C. for 60 minutes while stirring. 20 g BACTO-DEXTROSE (Trade name of a product manufactured and sold by the DIFCO laboratories) dissolved in 20 ml of distilled water, and the solution is passed through a Millipore sterilizing filter (Millipore Corporation, Bedford, Massachusetts, V.St.A.) from 0.45 micron filtered. The two preparations are combined and distributed in aliquots of 110 ml in 500 cm Roux flasks. '.

Production of the cultivation door:, · '

The strain Escherichia coli ATCC 21 972 is with sterile physiological Saline rehydrated and kept at 37 ° C for 18 hours incubated in Petri dishes, each containing 20 ml TRYPTOSE AGAR solid meclium by mixing 26 g of TRYPTOSE broth ■ and 30 g AGAR DIFCO (TRYPTÖGE broth and AGiIR DIFCO are from the Products manufactured and sold by DIFCO laboratories), replenishment with water up to a volume of 1 liter and 4-5 minutes

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Total heating of the mixture to 115 ° C is prepared.

A liquid culture medium (referred to as PP ^) is then prepared as described below: JOgProteose-Pepton Kr. 3 (a product manufactured and sold by the DIFCO laboratories), 4 g of yeast extract and 5% of glucose are dissolved in 1 liter of v / ater at 60 ⁰ C dissolved. After cooling, 5 g NaCl, 5 »O5 g HapHPCL and 1.2 g KH ₂ PO _{2 are} added. The medium, the pH of which is 6.9 to 7.0, is filtered through a Seitz EKii filter and distributed in 100 ml culture flasks.

The culture flasks in this way are inoculated with the smooth colonies obtained on the Petri dishes, one colony per 20 ml of liquid PP ₇ medium being used. The inoculated culture flasks are incubated for 6 hours at 37 ° C. while shaking on a pusher device (22 to 24 shots per minute).

Breeding of Sscherichia coli: · ■

2 Each of the 500 cm Eoux flasks containing the culture medium is mixed with an aliquot of 4 ail dex · _{Escher ichia coli culture obtained in the liquid PP 7} medium, ie with et v / a

4 χ Kk bacteria, inoculated. The flasks are shaking up Shaking devices with 22 to 24 shaking movements per minute incubated for one day at 37 ° C. Each cell culture is in 25 ml distilled water harvested, and crops from 15 roux flasks (one series) are combined in 1 liter flasks. A sample of 1 ml is taken from each series by means of a purity test.

For this purpose, 0.5 ml seeded onto TRYPTOoE agar broth in petri-3chalen and 7 Ta ^ e inkubierü at 54 ⁰ C. The remaining 0.5 ml

409823/113 4

236011B.

are sown in Petri dishes, the 20 ml Sabouraud dextrose ■ Agar obtained by dissolving 65 g of Sabouraud Dextrose Agar DIFCO (a manufactured and sold product) in 1 liter of hot distilled water and then sterilized. The culture will Incubated for 7 days at 22 ° C.

The harvests are ground with a sterile, aqueous percent neomycin sulfate solution (1.2 ml neomycin sulfate solution per 100 ml harvest) offset. The cells are exposed by sonicating the medium for 30 minutes in a Branson Europe sonicator model J 22 (Branson Europa N.Y., Soest, The Netherlands), whereby the medium is kept in a melting ice bath.

After the cells are broken, the suspension is used to separate the cell debris was centrifuged for 2 hours at 5 C and -2550 rpm. Of the The supernatant is first passed through a clarifying filter and then through an Ilillipore sterilizing filter (Hillipore. is a trademark of Killipore Corporation, Bedford, Massachusetts, V.St.A.) filtered "

The nitrate is treated with ammonium sulfate previously sterilized with ethylene oxide until 50 percent saturation (380 g of ammonium sulfate per liter of filtrate) added, and the mixture is 3 hours at Let stand room temperature. The precipitate obtained is centrifuged for 2 hours at 5 ° C. and 2550 rpm. The sediment will poured into a bag consisting mainly of regenerated cellulose and dialyzed against running water until the concentration of ammonium sulfate determined with Kessler's reagent has dropped to 0.1 to 0.01 percent.

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The enterotoxin preparation is sterilized by filtration through a Millipore sterilizing filter (Millipore is a Trademarks of Millipore Corporation) of 0.4-5 microns and freeze drying.

Example 2

An aliquot of 1 g of the freeze-dried enterotoxin preparation obtained in Example 1 is rehydrated by adding 20 ml of sterile, phosphate-buffered physiological saline solution (pH 7Λ). The sterile, phosphate-buffered physiological saline solution consists of 8 g KaCl, 2 g KCl, 11.5 g Na ₂ HrO ₄ , 2 g KH ₂ PO ₄ , 1.325 g CaCl ₂ χ 2H ₂ O, 1 g MgCl ₂ χ 6H ₂ O and 10 liters of distilled water.

The vaccine obtained in this way is thoroughly mixed under sterile conditions with 20 ml of sterile, complete Freund's adjuvant, ie with a water-in-oil emulsion of paraffin oil emulsified with Iannl monooleate and treated with killed Kyco.bacterium tuberculosis. The vaccines thus obtained is distributed in adjuvanshaltige I- ¹ ml ampoules, which were run down either zen or are sealed and contain a (LT) Enterotoxinrnenge corresponding to 100 mg of lyophilized preparation per vial. The content of each ampoule corresponds to a vaccine dosing unit. The vaccine is administered intramuscularly or subcutaneously to pregnant sows 3 to 6 per cent prior to piglets.

The adjuvant-containing vaccine can also be distributed in larger ampoules v / ground, whereby integer multiples of the dosage unit and thus the corresponding multiple ^ oscn vaccine preparations are obtained been.

4 0 9,823 / 1 1 34

B e i.'s ρ i e i 3

The process is carried out up to and including the 6-hour incubation of Escherichia coli AICG 21 972 -at 37 ° C in liquid PP medium carried out as described in Example 1.

The cultivation of Escherichia coli is then carried out in a liquid Growing medium carried out as described below:

6 ml aliquots of the Escherichia coli culture thus obtained (i.e., about 6 χ ίο "bacteria) are in 300 ml of liquid Culture flasks containing ΡΡ, medium are inoculated. The cultures are incubated for one day with shaking on shaking devices (22 to 24 shaking movements per minute). The harvests out 5 cultivation flasks (one series) are -united. Every harvest series is centrifuged for 2 hours at 2550 rpm, the sediment becomes'in Suspended 150 ml of distilled water, and a suspension sample is tested as in Example 1 for purity.

The cell suspension is sonicated for 30 minutes in a Branson Europe sonicator model J 22, the medium being held in a melting ice bath. After disruption of the cells, the suspension is centrifuged to separate the cell debris 2 hours at 5O ⁰ C and 2550 rpm. The supernatant is filtered through a 0.45 micron Millipore sterilizing filter to give 100 ml of filtrate. The pH of the filtrate is adjusted to 6.4 with η hydrochloric acid. Thiomersal is then added to the filtrate to a final concentration of 0.02 percent. .

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- OK -

Example 4

ml of a 2 percent aqueous solution of ALHYDRCGEL (one of SUPERFOS EXPORT Company manufactured and sold.) • Mix thoroughly with 80 ml of the filtrate obtained in Example 3 mixed. The adsorption of the enterotoxin on the ALHYiiROGSL is tested by means of the precipitation test with trichloroacetic acid.

20 ml of the supernatant are discarded. The rest of the mixture is distributed in ten 10 ml ampoules> which are locked. The content of each ampoule (9 nil) corresponds to one vaccine dosing unit of 100 rag (on a dry weight basis) of the heat labile (LT) Escherichia cöli enterotoxin.

The vaccine is given to the pregnant mother 3 to 6 weeks before administered intramuscularly or subcutaneously to the piglet. The adjuvant Vaccine - can also be distributed in larger ampoules, where integer multiples of the dosing unit and thus the corresponding Multi-dose vaccine preparations can be obtained.

Example 5

The procedure is carried out as in Example 3, but v / ob ei the mixture is distributed in 100 glass ampoules, which are sealed v / earth. The content of each ampoule (0.9 ml) corresponds to one vaccine dosage unit of 10 mg (on a dry weight basis) of the heat labile (LT) Escherichia coll Snterotoxin extract. The vaccine It is administered intramuscularly or subcutaneously to pregnant sows 3 to 6 weeks before piglet.

409823/1134

The prevention of piglet golibacillosis by subcutaneous administration of the heat-labile (LT) Escherichia according to the invention
coli enterotoxin preparations is shown by experiments I ₅ 2 and 3 below. In each of these experiments, the mother pigs are vaccinated 3 to 6 percent before piglet, and a mother pig is used as a control animal (LI) Escherichia coli enterotoxins infected using a collar tube for administration. In the case of three litters, some piglets are grounded - like from the
Tables I to III below can be seen - with 660 mg des
heat-stable (ST) Escherichia coli serotoxin obtained from strain ATGC 21 972 infected. The clinical development of the piglets is followed professionally, paying attention to the appearance of typical symptoms of colibacillosis.

Tn in Tables I to III means

Vaccine A the vaccine of Example 2 at the 100 mg (on

Dry weight basis) dosing unit,
Vaccine B the vaccine of Example 4 at the 100 mg (on

Dry weight basis) dosing unit and
Vaccine G the vaccine of Example 5 at the 10 mg (on

Dry weight basis) dosing unit.

(x) Hastitis appears 48 hours after piglet.
(xx) Weight loss 4 hours after the occurrence of the
Diarrhea. -

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Table I: Experiment 1

Mother pig vaccinated
with piglet No. infi
adorns
with Diarrhea ) except,
hours
• Die
lich
ability, Weight,
percent
(24 hours
after
Infection) 2-10 LT number of
hours
between
infection
and from
fracture _ _ no change No. 2-11 LT _ . - - + 3.5 2-12 LT - - - • + 9.5 A. 2-15 LT - - - no change 2-14- ST - - - + 3.6 2 2-15 ST - - - no change 2-16 ST - - - + 2.8 3-17 LT - - - + 10.6 3-18 LT - - + 11.8. 3-19 LT - - - + 6.3 A. 3-20 LT - - - + 5.8 3-21 ST - - - + 10 "3 3-22 ST - - - + 11.9 3-23 ST - - - + 10.4- 3-24- ST - - - + delete 10-01 LT - - 24 - - 10 10-02 LT 6th 24 - - 10.9 10-03 LT 6 to 12 12th - - 5.3 10-04- LT 6th 96 + - 14-, 7 - 10-05 LT 6th 10 - - 7.6 10-06 ST 6 to 12 - - no change fri ¹⁰ 10-07 ST - -3 - his change (Kon-
trolls) 10-08 ST 1 - - ce ine change 10-09 ST - 12th - no change 6th

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Table III Experiment 2

Mother pig vaccinated
with No. infi
adorns
with piglet JDauej}
Stun
the Die
lich
speed Weight _$
Percent,
(24 hours
after
Infection) - 47-12 LT Diarrhea no change No. 47-13 LT ' number of
hours
between
infection
and
outbreak ■ - - + 9 * 2 A. 47-14 LT - - no change 47-15 LT + 6 _S 4 47 47-16 LT - - + 6.6 48-08 LT - - " + • 5.2 B. 48-09 LT - «, + 6.7 48-10 LT «, - + 3.7 48 48-11 ¹ LT «. + 10.5 50-01 LT - ■ ■. 21 + - 19.8 50-02 LT - - - no change 50-03 LT - - + 2.6 - - 50-04 LT - 14-21 + - 22.1 50-05
50-06 .LT
LT - 48
10 r 18.8
- 3.2 50-07 LT 6th 21
I. - 28 _? 5 (Kon
trolls 14th
21 6th

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• 13th * ir . 21 5 vaccine
with • * * - Mr. : Experiment 3 'Piglet Farmer,
Stun
the ~ 14 - Die
lich
speed Weight,
percent
(24 hours
after
Infection) • Table III χ - B. 13-119 Diarrhea - 23601t8 --- - + 8.4 Mother pig 13-120 Number of
hours
between
infection
and from
fracture - - +. 11.4 13-121 infi
adorns
with .— ι - + 2 .1Tr. 13-122 LT - ■ - _ no change 13-123 LT - - - + 7.1 15th 13-124 LT - - - 2 - + 8.1 13-125 LT - - - + 8 13-126 LT 4th ■ ~ " - + 5.8 13-127 LT - ; 40 + - 21st 13-128 LT "* · : - - + 8.4. σ 21-110 LT 8th 48 + - 15 21-111 LT ■ 6-11 - "6.9 20th 21-112 LT 48 + - l *, 3 {Kon-,
trolls 21-114 LT 3 ■ - i - - - .- 5.9 21-115 LT 4th 6th - - 6.3 ■ 21-116 LT '■ - - - 3.6 σ 21-117 LT . 6th - - - 2 21-118 LT - - - - 12.5 15-129 LT ' - ι - - + 7.4 15-130 LT 6 · - - + 4.9 15-131 LT - - + 13.2 15-132 LT - - - + 16 15-133 LT - - -. no change 20-101 LT - 6th - 3.6 20-102 LT - 6th - - 16.5 20-103 LT - 5th 4th - - 4.5 XX, 20-104 LT 6th 6th - - 5.3 ? 0-105 LT 25th 6th - - 4.7 0-106
0-107 LT 19th 4-9 - No change,
- 3.2 0-108 LT j 6th '6 - ■ - 5i7 0-109 LT 3 6th - - 4.2 LT
LT 6th LT 6th LT

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The results of the experiments show that 6 out of 7 litters from vaccinated mother pigs are protected against oral infection with Escherichia coli enterotoxins. The protection achieved is 90 percent for one litter and 100 percent for the 5 other litters. No liurf derived from the control pigs is resistant to the same infection with enterotoxins; the mortality and morbidity among the control piglets is 71.4 to 88 ₅ 8 percent.

In five out of seven litters of vaccinated mother pigs are all piglets fully protected; in a litter 8 out of 10 piglets are completely protected. For those with IT enterotoxin infected piglets and piglets derived from control pigs obtained the following results: In a litter, 5 out of 5 animals show in the second litter 5 of 7 animals and in the third VJurf 8 of> 9 animals typical symptoms.

Example?

A 12 g aliquot of that prepared according to Example 1 and freeze-dried ent erot oxinpr Spar at s v / ird in 1 liter pyrogen-free distilled water suspended. The suspension is mixed with a solution of 20 g of ALHYDROGEL (a from SÜPERF08 EXPORT Company) mixed in 1 liter of pyrogen-free distilled water. The pH value is adjusted to 6 with η hydrochloric acid, and the mixture v / ird · for adsorption of the enterotoxin on ALHYDROGEL 40 hours in Allowed to stand in the dark at seam temperature, stirring Mn and again. The final pH is 6.3.

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After centrifugation, the supernatant is discarded and the sediment is removed by adding 2.2 liters of a 5O: 5O mixture of physiological serum and buffer (22 g Na ₂ HPO ₄ (m / 10) and 78 g KH ₂ PO ₄ (m / 10) and 10 mg thiomersal rehydrated to 100 ml \ pH 6.3). The mixture is ^{stirred at A-0} G for 2 hours and divided into two 1100 ml batches, labeled A and B. Batch A contains 5 »4 · mg of Enterotoxinpräparats and 9 mg AI hydrogel pro.ml and is divided in ^m l aliquots of 5, which thus contained 27 mg of Enterotoxinpräparats. The aliquots are "filled into glass ampoules, which are then melted off. Batch B is diluted by adding 303 ml of a 50: 50 mixture of physiological serum and buffer (22 g Na ₂ HPO ₄ (m / 10) and 78 g KH ₂ PO ₄ (m / 10) and 10 mg thiomersal to 100 ml; pH 6.3) to which 2.7 g of ALHYDROGEL was added. Gharge B contains 4-, 3 mg of the enterotoxin ^{preparation and 9 1} ^ g of ALHYDROGEL per ' ml and are divided into aliquots of 2.5 ml, which thus contain 10.75 * ng of the enterotoxin preparation. The aliquots are filled into glass ampoules, which are then sealed off.

The vaccine preparations made from batches A and B are used as described below in breeding centers infested with diarrhea caused by coliform bacteria, on sows checked. For this purpose, the sows are subcutaneously administered a single dose of vaccine 3 to 6 weeks before the piglets vaccinated: 59 sows received a dose of the batch A vaccine and 28 dams a dose of the Batch B vaccine. 50 sows serve as controls and receive the same Volumes of the same ALHYDROGEL buffer mixture without the enteric

409823/1134

t oxinext r act ·,

All piglets are observed for the occurrence of diarrhea: tet. On and after the second day of the clinical symptom of the disease Merkel are treated daily with chloramphenicol, ampicillin or sulfadoxin / trimethoprim. The results are in Table IV taking into account the consistency of the faeces, the duration of the diarrhea, the number of administrations of antibiotics and dehydration mortality.

ν ■

Table IV

Vaccine-
dose number
the
Throws Throw with
Di £
number heavier
irrhoe (*)
percent Throws with S
number mortality
percent 5 ml of
Batch A 59 15th 25.4 2 3.5 2.5 ml
from
Batch B 28 10 55.7 2 7.1 placebo 50 24 48 8th *
16

(x) Liquid faeces for more than two days, multiple administrations of antibiotics, with and without mortality

(xx) 25 sows receive 2.5 ml. and the others 25 motherships / one 5 ml.

The results compiled in Table IV show that the administration of vaccines according to the invention of the disease and Mortality percentage is considerably reduced., With the administration of the 27 mg enterotoxin dosage unit better protection is obtained for the sows.

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Claims (6)

Claims 23601 Ί 8 φ, vaccines against piglet coliballosis, containing "an effective Amount of cell-free, heat-labile (LT) Escherichia coli enterotoxin and an adjuvant. 2. Vaccines according to claim 1, characterized in that the liitzelabile (LT) Escherichia coli enterotoxin is a cell-free and essentially ναμ lipopolysaccharide-free extract from Escherichia coli. 3. Vaccines according to claim 1 and 2, characterized in that the heat-labile (LT) Escherichia coli enterotoxin from the strain Escherichia coli A ¹ SGG 21,972 originates. 4-, vaccines according to claim 1 to 3> characterized in that the adjuvant is an oily adjuvant or · a gelatinous aluminum compound indung is. 5. Vaccines according to claim 1 to 4-, characterized in that the enterotoxin bosi unit 10 to 150 mg "freeze-dried Extract amounts. 6. A method for producing the vaccines according to claim 1 to 5 » characterized in that the heat-labile is obtained from cells of a heat-labile enterotoxin producing Escherichia coli-Staraiaes (LT) Escherichia coli enterotoxin isolated and mixed with an adjuvant, ■ / 409823/1134